1. Field of the Invention
The present invention relates to a method for amplification of RNA, especially to a method for synthesis of RNA by means of a reverse transcriptase-polymerase chain reaction (hereinafter abbreviated as RT-PCR).
Regarding expression of substances in the present specification, the singular form of a substance includes both singular and plural.
2. Description of the Related Art
An RT-PCR method is a procedure in which an RNA is converted into a complementary DNA (cDNA) using a reverse transcriptase and then the cDNA is amplified by a PCR method. Since an RT-PCR method enables a quantitative analysis even with a trace amount of the RNA, it is regarded currently as an analytical method having the highest sensitivity which is essential for a detection of an virus having an RNA as a gene, for a quantitative detection of an mRNA, for an analysis of an expressed gene by a base sequencing, and for an analysis and a production of an expression product by a cDNA cloning.
A PCR method which is conducted subsequently to an RT reaction in an RT-PCR method is a procedure capable of amplifying an intended DNA fragment by several hundred thousand times by repeating a DNA synthesis reaction between the primers sandwiching a certain region of a DNA chain. A PCR method is disclosed in Japanese Laid-open Patent Publication No. S61-274697 which is an invention by Mullis et al.
Another method for amplifying an RNA is an NASBA (Nucleic Acid Sequence Based Amplification) method etc. has been developed. Since this method allows an amplification reaction to be performed directly from an RNA, it is suitable in a study employing an RNA as a template, and thus being brought into use recently.
Furthermore, since an NASBA method involves no denaturation process such as a PCR, it needs no thermal cycles and thus can effect an amplification characteristically at a constant temperature.
However, since any of the RNA amplification methods including those described above is based on an enzymatic reaction, it is well known to allow the reaction to be inhibited potently by pigments, proteins, saccharides or unknown impurities contained in a biological sample.
Accordingly, a process, prior to an RNA amplification described above, for separating a cell, a fungus, a bacterium and a virus etc. (hereinafter referred to as an RNA inclusion body) from a sample followed by extracting an RNA from such RNA inclusion body is required prior to the nucleic acid synthesis described above. Such process has conventionally been a procedure in which a biological sample is treated with an enzyme, a surfactant or a chaotropic agent, etc. and then an RNA is extracted with phenol or phenol/chloroform, etc. Recently, an ion exchange resin, a glass filter, a glass bead or a reagent having a protein coagulating effect is employed in a process for extracting an RNA.
Nevertheless, since any of the methods described above poses a difficulty in removing the impurities completely when employed for purifying an RNA in a sample and frequently fails to recover a nucleic acid in a sample at a constant yield, it fails to allow a subsequent RNA amplification to take place successfully especially when the content of intended nucleic acids in a sample is low. Furthermore, any of these purification methods involves a complicated, time-consuming operation as well as a higher possibility of a contamination during the operation.
Also since an RNA is always at a risk of a degradation by an RNA degrading enzyme (RNase) which is present generally in all biological samples, and it is required to inactivate the RNase rapidly upon a purification and to keep strict operation and control also during and after the purification to ensure no contamination with an RNase.
An object of the present invention is to provide a novel reaction solution capable of amplifying an RNA in a biological sample efficiently while suppressing a substance having an inhibitory effect on a nucleic acid synthesis, whereby providing a method for amplifying said RNA in said sample by adding said sample or an RNA inclusion body in said sample directly to said reaction solution.
Thus, the present invention is a method for amplification of RNA comprising adding a sample directly to a reaction solution to conduct an RNA amplification reaction whereby amplifying the RNA present in the sample immediately. In this method, a sample may be added to a reaction solution, or, alternatively the reaction solution is added to the sample, thus no order of the addition being specified.
While an RT-PCR method are included in an RNA amplification method in the present invention, they are not limitative and any other methods utilizing enzymatic reactions to amplify an RNA may also be employed.
In the present invention, the expression xe2x80x9cadding directlyxe2x80x9d means that xe2x80x9cafter a cell, a fungus, a bacterium and a virus etc. (hereinafter referred to as an RNA inclusion body) are separated from a sample prior to an RNA amplificaiton, there is no need of a process for extracting an RNA from such RNA inclusion bodyxe2x80x9d.
In the present invention, a sample means an RNA inclusion body in a biological sample or a biological sample itself. A biological sample means an animal or plant tissue, a body fluid, an excreted matter and the like, and an RNA inclusion body means a cell, a fungus, a virus and the like. A body fluid includes a blood-derived sample such as blood, plasma and serum as well as spinal fluid, saliva and milk and the like. An excretion includes urine, feces and the like. A cell may be, but not limited to, a leukocyte separated from a blood or a spinal fluid, as well as an buccal mucosal cell and the like.
The present invention is a method for amplification of RNA described above wherein the pH of said reaction solution for a gene amplification at 25xc2x0 C. is 8.2 or higher, that at 55xc2x0 C. is 7.4 or higher, and/or that at 70xc2x0 C. is 7.1 or higher.
The present invention is a method for amplification of RNA described above wherein a polyamine is added to said reaction system.
The present invention is a method for amplification of RNA described above wherein a sulfated polysaccharide and/or salts thereof (hereinafter referred to together as a sulfated polysaccharide) is added to said reaction system.
The present invention is a method for amplification of RNA described above wherein the pH of said reaction solution for a gene amplification at 25xc2x0 C. is 8.2 or higher, that at 55xc2x0 C. is 7.4 or higher, and/or that at 70xc2x0 C. is 7.1 or higher; and/or at least one of a polyamine, a sulfated polysaccharide and/or salts thereof and dithiothreitol are added to said reaction system.
The present invention allows a biologically-derived sample to be added directly to a reaction solution to effect a direct amplification of an RNA which is present in the sample. Thus, the invention enables a convenient, rapid and highly sensitive detection of a foreign organism such as an RNA virus including hepatitis C virus (HCV) existing latently in a biologically-derived sample, and a retrovirus including a human immunodeficiency virus (HIV) as well as a variant cell such as a carcinoma cell existing as a cell which is now quite distinct from a host cell. The invention also allows a detection of an mRNA which is subjected to an intracellular transcription, an analysis of an expression gene by a base sequencing and an analysis and a production of an expression product by a cDNA cloning to be performed conveniently and rapidly. Furthermore, the invention which employs a direct amplification of an RNA from a biologically-derived sample can avoid the effect of an RNA degradation by an RNase which has conventionally been experienced during an extraction and a purification of an RNA.